The present invention relates to an assay method for detecting potential for or propensity to cardiovascular disease (CVD) in a subject, e.g. a human or non-human animal, especially a mammal, and in particular to an assay method which may be used to detect a potential for CVD or a propensity to CVD before the onset of CVD symptoms noticeable by the subject.
CVD is a major source of ill health among the human population. In 1998 approximately 40% of all deaths in the western world was a result of CVD (i.e. 1 in every 2.5 deaths). For 2002, it is estimated that, in the USA, over one million people will suffer from a new or recurrent-coronary attack, and more than 40% of the people suffering from these attacks will die. Many of these people will die suddenly without ever having been hospitalised or treated. Many will not have realised that they were susceptible to CVD.
Early or pre-emptive treatment such as a change of diet, reduction or cessation of smoking, increase in exercise, reduction of body weight, etc., has, however, a high success rate of preventing CVD or reducing the propensity to CVD. Thus if CVD or potential for or propensity to CVD can be detected, effective treatment is available.
There is accordingly a need for methods which can be used to detect CVD and especially, the potential for or propensity to CVD, before the disease has progressed beyond the stage where treatment (e.g. change of life style and/or habit) is routinely successful. In particular, there is a need for methods which can be used to detect CVD at the early stages when the symptoms are not apparent to a subject or to a third party, e.g. a physician, i.e. methods for testing “symptom-free” subjects are required.
Such methods may be used to screen the general population (i.e. in mass screening) or at-risk groups within the population, e.g. males over 40, workers in high stress jobs, individuals with unhealthy diets, individuals suffering from clinical obesity, smokers, etc. In cases where potential for CVD or propensity to CVD is diagnosed, pre-emptive treatment may be given and/or the patient may be encouraged to make adjustments to lifestyle and habit.
Likewise, where potential for CVD or propensity to CVD is detected, a patient may be submitted to further testing, e.g. using more expensive or time consuming techniques, such as ECG, with and without physical activity, radioisotope imaging of myocardial perfusion, X-ray (e.g. CT) myocardial angiography, MR myocardial angiography or perfusion imaging, etc. Thus by confirming the possible presence of, or potential for, or propensity to, CVD by using the cheap and facile assay method of the invention in an initial screen (e.g. in a mass screen of “symptom-free” healthy subjects) the likelihood of detecting or identifying undiscovered CVD, or potential for CVD, before health damage becomes irreversible is increased whilst, at the same time, unnecessary use of expensive and time-consuming tests is limited.
By “CVD” is meant any condition of the heart, arteries, or veins which disrupts the supply of oxygen to life-sustaining areas of the body such as the brain, the heart etc. Examples of CVDs are arteriosclerosis, acute myocardial infarction, angina pectoris, ischemic heart disease, cerebrovascular disease, stroke, subarachnoid haemorrhage, intra-cerebral haemorrhage, cerebral infarction, congestive heart failure, angina, heart attack, cardiac arrest and arrhythmia.
The present invention is based on the surprising finding that the protein calprotectin is a useful “marker” or “indicator” of potential for CVD or propensity to CVD before the onset of CVD symptoms (i.e. in symptom-free subjects). In particular it has been surprisingly found that abnormally high calprotectin levels in various body fluids is indicative of susceptibility to CVD before the onset of CVD symptoms is apparent to a subject or to a third party (e.g. a physician).
For the avoidance of doubt, the term calprotectin is used herein synonymously with “L1 protein”, “MRP 8/14”, “cystic fibrosis (associated) antigen (CFA)” and “calgranulin”.
Calprotectin exists in both dimeric and trimeric forms. As a dimer, calprotectin comprises the polypeptide chains S100A8 and S100A9. As a trimer, calprotectin is a 36 kDa heterotrimeric protein with two heavy (14 kD) and one light chain (8 kD) non-covalently linked.
Calprotectin is a calcium binding protein and when bound to calcium, calprotectin is resistant to heat and to proteolysis. This may allow for a wide range of assay techniques and conditions to be employed.
Epitope mapping of calprotectin shows that antibodies with specificity for the complex and/or its single protein chains may be produced. At least four separate immunogenic sites have been shown to exist on the calprotectin complex. Some antibodies recognise either the heavy or the light chain, whilst others recognise both.
Calprotectin is found in cells, tissues and fluids in all parts of the human body and is derived predominantly from neutrophils and monocytes. Calprotectin is probably present in all individuals since amongst more than 5,000 individuals tested, no calprotectin free individual was found. Calprotectin is also found in rats, mice, rabbits, sheep, cattle and pigs. It is therefore an abundant ubiquitous molecule.
In vivo, calprotectin is involved in numerous biological functions including intracellular signal transduction, neutrophil activation, inhibition of intracellular enzymes involved in cell proliferation, antimicrobial activity and in neutrophil defence. Calprotectin is also a regulatory protein in inflammatory reactions and in this role may function to stimulate immunoglobulin production, chemotactic factor activity and neutrophil immobilising factor.
Whilst body fluids probably always comprise calprotectin, the concentration of calprotectin in various body fluids has been found to change, for example, to increase, in a number of disease conditions (e.g. inflammatory, infectious and malignant diseases). Thus measurement of the concentration of calprotectin in body fluid from patients suffering from such disease conditions (i.e. in individuals showing symptoms noticeable to the subject and/or to a third party) and comparing the calprotectin concentration determined to that in body fluid from, for example, a healthy (i.e. a non-diseased) subject may be used as a means of diagnosing such diseases.
For example, whilst the symptoms of bacterial and viral infections are very similar and diagnosis from their symptoms alone may be difficult, the concentration of calprotectin in the plasma/serum of the infected subject increases approximately 1 to 2 times with viral infections but around 1 to 18 times with bacterial infections. Thus the subject having noticed the symptoms of infection, can have the concentration of calprotectin in their body fluid measured and their infection diagnosed and treated accordingly.
Other diseases in which calprotectin may be used as a diagnostic test include: rheumatic diseases (e.g. rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus), Sjøgrens syndrome, intraocular inflammatory conditions, cystic fibrosis, acute and chronic lung disease, lung carcinoma (squamous cells), pulmonary cancers, colorectal cancer, inflammatory bowel disease, gastric cancer, colorectal adenoma or cancer, Chrohn's disease, ulcerative colitis, gastrointestinal mucosal inflammation, urinary stones, alcoholic liver disease, oral inflammatory mucosal disease, CNS inflammatory disease (e.g. multiple sclerosis and acute encephalitis), HIV infection, secondary CNS infections in HIV infected patients, urinary tract infections, cystitis, pyelonephritis, endogenous posterior uveitis, haematological conditions (e.g. leukaemia), febrile conditions (infectious and non-infectious), acute myocardial infarction and apheresis.
The plasma concentration of calprotectin has also been found to increase during open heart surgery (Semb, A. G. et al, Eur. J. Cardio-thorac Surg. (1991) 5:363-367, Saatvedt, K. et al., Scand. J. Thor. Cardiovasc. Surg. (1996) 30: 53-60, Moen, O. et al., Perfusion (1994) 9:109-117). More specifically, Saatvedt et al. report that calprotectin concentration rises after the start of cardiopulmonary bypass and peaks 48 hours post-operatively.
It has now surprisingly been found that the potential for CVD or propensity to CVD in a subject can be assessed by determining the concentration of calprotectin in a calprotectin-containing sample taken from said subject. In other words, it has been found that determination of the concentration of calprotectin in a calprotectin-containing sample taken from a subject can be used to predict, prior to the onset of symptoms which are noticeable to the subject or to a third party (e.g. a physician) whether or how likely the subject is to suffer CVD.
By “potential for” or “propensity to” is meant the likelihood or probability that the currently symptom-free subject being tested will suffer CVD in the future. This might take the form of an index, ratio, percentage or similar number reflective of the relative risk of CVD in the future (e.g. in the following 1-2 years, at least in the following 6 months).